Vascular endothelial growth factor--structure and functions

Vascular endothelial cell growth factor (VEGF), originally described as a vascular permeability factor, is currently known as one of the main factors which regulate angiogenesis. It plays an important role in the regulation of normal as well as pathological angiogenesis. In this paper we try to shortly review the actual knowledge on VEGF protein family, its expression, VEGF receptors and role of VEGF in signal transduction. The aim of this review is also to summarize recent achievements in research on biological functions of vascular endothelial growth factor and their clinical applications.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.     

Characterization of vascular endothelial growth factor receptors on the endothelial cell surface during hypoxia using whole cell binding arrays

Angiogenesis plays a central role in a variety of important biological processes such as reproduction, tissue development, and wound healing, as well as being critical to tumor formation in cancer. The development of chromosomal substitution (consomic) rat strains has permitted the chromosomal localization of genetic factors critical to angiogenesis, but many questions remain as to the mechanisms involved. Here we utilize a novel cell capture assay to assess changes in the functional expression of vascular endothelial growth factor (VEGF) receptors on the surface of vascular endothelial cells isolated from rat strains that are normal or impaired in angiogenesis. We show that functional VEGF receptor expression is increased under hypoxic conditions in rat strains that exhibit normal angiogenesis but not in a strain impaired in angiogenesis. This result implicates the dysregulation of VEGF receptor expression levels on the endothelial cell surface as a key factor in impaired angiogenesis.     

Constitutive and inducible expression and regulation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), which was originally discovered as vascular permeability factor, is critical to human cancer angiogenesis through its potent functions as a stimulator of endothelial cell survival, mitogenesis, migration, differentiation and self-assembly, as well as vascular permeability, immunosuppression and mobilization of endothelial progenitor cells from the bone marrow into the peripheral circulation. Genetic alterations and a chaotic tumor microenvironment, such as hypoxia, acidosis, free radicals, and cytokines, are clearly attributed to numerous abnormalities in the expression and signaling of VEGF and its receptors. These perturbations confer a tremendous survival and growth advantage to vascular endothelial cells as manifested by exuberant tumor angiogenesis and a consequent malignant phenotype. Understanding the regulatory mechanisms of both inducible and constitutive VEGF expression will be crucial in designing effective therapeutic strategies targeting VEGF to control tumor growth and metastasis. In this review, molecular regulation of VEGF expression in tumor cells is discussed.     

Vascular endothelial growth factor receptor-2 in breast cancer

Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR was structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival.     

血管内皮生长因子受体信号转导通路与肿瘤血管生成

血管内皮生长因子是促进血管生成的重要调节因子.它能促进内皮细胞增殖、迁移,阻止内皮细胞凋亡、管腔网状结构退化,增加血管渗透性.所有这些作用都是通过血管内皮生长因子受体信号转导通路实现的.它们在肿瘤血管生成、肿瘤生长中起着重要的作用.以血管内皮生长因子受体信号转导通路为靶点是开发肿瘤血管生成抑制剂的理想策略.     

Vascular permeability factor: a unique regulator of blood vessel function.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent polypeptide regulator of blood vessel function. VPF promotes an array of responses in endothelium, including hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VPF regulates the expression of tissue factor and the glucose transporter. All of the endothelial cell responses to VPF are evidently mediated by high affinity cell surface receptors. Thus, endothelial cells have a unique and specific spectrum of responses to VPF. Since each of the responses of endothelial cells to VPF are also elicited by agonists, such as bFGF, TNF, histamine and others, it remains a major challenge to determine how post-receptor signalling pathways maintain both specificity and redundancy in cellular responses to various agonists.     

Autocrine VEGF signalling on M2 macrophages regulates PD‐L1 expression for immunomodulation of T cells

M2‐polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death‐ligand 1 (PD‐L1) on M2 macrophages was significantly up‐regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD‐L1 expression on M2 macrophages was dramatically down‐regulated. Furthermore, transplantation of PD‐L1⁺ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4⁺/CD8⁺ T cells in the peripheral blood and increased CD4⁺CD25⁺ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD‐1 signalling blockade in cancer therapy.     

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

Anti‐angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion‐associated domain in MUC4 and other proteins (AMOP) domain at the C‐terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C‐terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)‐basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF‐stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase‐dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen‐tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.     

血管内皮细胞生长因子在非血管新生方面的重要功能

血管内皮细胞生长因子(vascular endothelial growth factor,VEGF或VEGF-A),又称为血管通透因子(vascular permeable factor,VPF)是一种具有多种功能的生物大分子,它是分泌性糖蛋白生长因子超家族中的一员.VEGF主要通过两个高亲和力的酪氨酸激酶受体来传递各种信号:VEGF受体1和2(VEGFR1,VEGFR2),从而引起细胞的多种生理反应.在胚胎时期,VEGF可以促进血管内皮细胞的增殖、迁移、管状形成和提高内皮细胞的存活率,对于血管新生和发育十分关键;而在成体时期,VEGF则主要参与正常血管结构的维持,并调节生理和病理性血管新生.近几年来的临床试验表明,使用多种阻断VEGF作用的抑制剂能有效促进肿瘤血管的退化和减小肿瘤的体积,但是同时在部分病人中也观察到了多方面的副作用.这些结果显示,VEGF也具有非血管新生方面的重要功能.因此,在研制基于拮抗VEGF作用的抗癌药物时,这些功能更不容忽视.研究表明,在成体的小肠、胰岛、甲状腺、肾脏和肝脏等器官组织中,VEGF都发挥着十分重要的作用,如果VEGF水平降低,这些器官组织的毛细血管网状结构将部分退化.VEGF还可以促进骨髓形成、组织修复与再生、促进卵巢囊泡成熟,并且参与血栓、炎症反应和缺氧缺血的病理过程.本文主要对VEGF在血管新生之外的功能及其分子机制进行了简要探讨.     

Fibroblast Growth Factor-2 (FGF-2) Induces Vascular Endothelial Growth Factor (VEGF) Expression in the Endothelial Cells of Forming Capillaries: An Autocrine Mechanism Contributing to Angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2–induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-M_r FGF-2 (22–24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

Vascular endothelial growth factor in astroglioma stem cell biology and response to therapy

Malignant astrogliomas are among the most aggressive, highly vascular and infiltrating tumours bearing a dismal prognosis, mainly due to their resistance to current radiation treatment and chemotherapy. Efforts to identify and target the mechanisms that underlie astroglioma resistance have recently focused on candidate cancer stem cells, their biological properties, interplay with their local microenvironment or 'niche', and their role in tumour progression and recurrence. Both paracrine and autocrine regulation of astroglioma cell behaviour by locally produced cytokines such as the vascular endothelial growth factor (VEGF) are emerging as key factors that determine astroglioma cell fate. Here, we review these recent rapid advances in astroglioma research, with emphasis on the significance of VEGF in astroglioma stem-like cell biology. Furthermore, we highlight the unique DNA damage checkpoint properties of the CD133-marker-positive astroglioma stem-like cells, discuss their potential involvement in astroglioma radioresistance, and consider the implications of this new knowledge for designing combinatorial, more efficient therapeutic strategies.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

促血管生成素的生物学特点和应用前景

促血管生成素（Ang）家族是调节血管生成的一类细胞因子,包括Ang-1、Ang-2、Ang-3和Ang-4等4个成员,Ang-1和Ang-2是其中最重要的成员。Tie-2是Ang家族的共同受体。Ang-1与Tie-2结合后激活下游信号通路,起到抑制内皮凋亡、促进内皮存活和迁移、维持血管完整性的作用;Ang-2则是Ang-1天然的抑制剂,其拮抗的效应与局部血管内皮生长因子（VEGF）的水平有关,VEGF存在时促进新生血管形成,VEGF缺乏时则有利于血管的消退。Ang参与生理和病理性的血管新生,与肿瘤和其他疾病有密切的关系,有广泛的应用前景。     

Vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. Its activity is mediated by the high affinity tyrosine kinase receptors, KDR/Flk-1 and Flt-1. In this article, recently discovered structural, molecular and biological properties of VEGF are described. Among the topics discussed are VEGF and VEGF receptor structure and bioactivity, the regulation of VEGF expression, the role of VEGF and its receptors in vascular development, and the involvement of VEGF and its receptors in normal and pathological (ocular and tumor) angiogenesis.     

The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor

Angiogenesis has an essential role in many important pathological and physiological settings. It has been shown that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent cytokine expressed by most malignant tumors, has critical roles in vasculogenesis and both physiological and pathological angiogenesis. We report here that at non-toxic levels, the neurotransmitter dopamine strongly and selectively inhibited the vascular permeabilizing and angiogenic activities of VPF/VEGF. Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VPF/VEGF binding, receptor phosphorylation and subsequent signaling steps. The action of dopamine was specific for VPF/VEGF and did not affect other mediators of microvascular permeability or endothelial-cell proliferation or migration. These results reveal a new link between the nervous system and angiogenesis and indicate that dopamine and other D2 receptors, already in clinical use for other purposes, might have value in anti-angiogenesis therapy.     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

Mechanisms of angiogenesis

Tissue activity of angiogenesis depends on the balance of many stimulating or inhibiting factors. The key signaling system that regulates proliferation and migration of endothelial cells forming the basis of any vessel are vascular endothelium growth factors (VEGF) and their receptors. The VEGF-dependent signaling system is necessary for formation of the embryonic vascular system. Neoangiogenesis during tumor growth is also associated with activation of this signaling system. The biological significance of the effect of such system on the cells depends on the content in tissue of various factors of the VEGF family and their receptors, while in the case of VEGFA it is defined by the ratio of different isoforms of this growth factor. A number of other signaling systems are also involved in regulation of the main steps of vessel formation. The signaling system Dll4/Notch regulates selection of endothelial cells for beginning of angiogenic expansion by endowing particular properties to endothelial cells leading in this process. An important step in vessel stabilization and maturation is vascular wall formation. Signaling system PDGFB/PDGFRbeta as well as angiopoietins Ang1, Ang2, and their receptor Tie2 are involved in recruiting mural cells (pericytes and smooth muscle cells). Identification of key molecules involved in the regulation of angiogenesis may provide new possibilities for development of drugs suitable for inhibition of angiogenesis or its stimulation in various pathologies.     

Differential roles of VEGF: Relevance to tissue fibrosis

Excessive extracellular matrix deposition and pathological vascularization are characteristics of fibrosis, which compromises the normal functioning of organs. Although whether angiogenesis can be induced and can occur in parallel with the progression of fibrosis has not been definitely determined, angiogenesis undoubtedly plays a vital role in fibrosis. Since vascular endothelial growth factor (VEGF) is one of the most effective proangiogenic factors, VEGF-targeting interventions have been a focus for the development of therapeutic strategies against fibrosis. In this review, we will summarize the current knowledge of the role of VEGF and its relevant mechanisms in fibrotic biology. We especially expect to provide a comprehensive overview of the therapeutic potential of VEGF-targeted therapy strategies to restore vascular function in the organs affected by fibrosis.     

Manipulation of ovarian follicle development by injecting vascular endothelial growth factor (VEGF) gene

The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation in the ovary. Our recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the thecal angiogenesis during follicular development. In this review, we focus on the vasculature and the expression of angiogenic factors during follicular development in a mammalian ovary.